Aberrant Double-Strand Break Repair in rad51 Mutants of Saccharomyces cerevisiae (original) (raw)
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Molecular and cellular biology, 1995
HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove U...
Suppression of the Double-Strand-Break-Repair Defect of the Saccharomyces cerevisiae rad57 Mutant
Genetics, 2009
The Rad51 paralogs Rad55 and Rad57 form a heterodimer required to mediate the formation and/or stabilization of the Rad51 filament. To further characterize the function of Rad55-Rad57, we used a combination of rad57 partial suppressors to determine whether the DNA repair and recombination defects of the rad57 mutant could be completely suppressed. The combination of all suppressors, elevated temperature, srs2, rad51-I345T, and mating-type (MAT) heterozygosity resulted in almost complete suppression of the rad57 mutant defect in the recruitment of Rad51 to DNA-damaged sites, as well as survival in response to ionizing radiation and camptothecin. In a physical assay to monitor the kinetics of double-strand-break (DSB)-induced gene conversion, the rad57 mutant defect was effectively suppressed by srs2 and MAT heterozygosity, but these same suppressors failed to suppress the spontaneous recombination defect. Thus the Rad55-Rad57 heterodimer appears to have a unique function in spontaneo...
Recruitment of the Recombinational Repair Machinery to a DNA Double-Strand Break in Yeast
Molecular Cell, 2003
viewed in Pâ ques and Haber, 1999; Sung et al., 2000). In the mouse, a homozygous null allele of RAD51 leads to embryonic lethality (Tsuzuki et al., 1996), and muta-Program in Molecular Medicine tions in RAD genes are associated with a spectrum of University of Massachusetts Medical School diseases, including cancer (reviewed in Ivanov and Ha-Worcester, Massachusetts 01605 ber, 1997; Jasin, 2000; Michelson and Weinert, 2000). 2 Institute of Biotechnology and Studies in yeast have suggested a sequence of molec-Department of Molecular Medicine ular events that occur following formation of a DSB (re-. First, the 5Ј ends of DNA that flank San Antonio, Texas 78245 the break are resected by an exonuclease. Rad51p, a functional homolog of the E. coli RecA recombinase, then binds the exposed single-stranded tails forming a right-Summary handed helical nucleoprotein filament. In vitro, Rad52p (Sung, 1997a) and a Rad55p/Rad57p heterodimer (Sung, Repair of DNA double-strand breaks (DSBs) by homol-1997b) can promote this early step by overcoming the ogous recombination requires members of the RAD52 inhibitory effects of the heterotrimeric single-stranded epistasis group. Here we use chromatin immunopre-DNA binding protein, RPA. The Rad51p nucleoprotein cipitation (ChIP) to examine the temporal order of filament is then believed to function in cooperation with recruitment of Rad51p, Rad52p, Rad54p, Rad55p, Rad54p to search the genome for a homologous pairing and RPA to a single, induced DSB in yeast. Our results partner and to form a heteroduplex "joint molecule" (Petsuggest a sequential, interdependent assembly of ukhova et al., 1998, 2000). Joint molecule formation is Rad proteins adjacent to the DSB initiated by binding followed by extension of the incoming strand by DNA of Rad51p. ChIP time courses from various mutant polymerases and branch migration, ultimately leading strains and additional biochemical studies suggest to restoration of the genetic information spanning the that Rad52p, Rad55p, and Rad54p each help promote break (reviewed in Pâ ques and Haber, 1999). the formation and/or stabilization of the Rad51p nu-Much less is known about how Rad proteins functioncleoprotein filament. We also find that all four Rad ally cooperate during DSB repair in vivo. Immunofluoresproteins associate with homologous donor sequences cence studies have shown that Rad51p, Rad52p, and during strand invasion. These studies provide a near Rad54p colocalize to "foci" in response to DNA damage comprehensive view of the molecular events required in vivo (Haaf et al., 1995; Tan et al., 1999), suggesting for the in vivo assembly of a functional Rad51p presynthat Rad proteins might function together within a larger, aptic filament. multiprotein complex. Consistent with this view, coimmunoprecipitation and yeast two-hybrid assays have Introduction shown that many members of the RAD52 group can interact with each other (Golub et al., 1997; Hays et al., DNA double-strand breaks (DSBs) arise in DNA due to 1995; Johnson and Symington, 1995; Krejci et al., 2001). environmental insults such as ionizing radiation or In contrast, recent studies indicate that the composition chemical exposure. DSBs also play an important role as of the damage-induced foci are dynamic, and photointermediates in DNA replication, immunoglobulin V(D)J bleaching studies indicate that several Rad proteins recombination, meiotic and mitotic crossing-over, and have very different diffusion coefficients, suggesting that yeast mating-type switching. Failure to correctly prothey may not exist together in a preassembled protein cess these DSBs can result in deletion or insertion of complex (Essers et al., 2002). genetic information, chromosomal fragmentation, trans-We wished to dissect how Rad proteins are recruited location, and chromosome loss. and function at a DSB in vivo. Here we use chromatin Homologous recombination (HR) is a major pathway immunoprecipitation (ChIP) analyses to examine the of DSB repair in all eukaryotes and has a distinct advantemporal order of Rad protein recruitment to a single, tage over other mechanisms in that it is mostly error induced DSB in yeast. Our results suggest a sequential free. Repair of DSBs by HR requires the RAD52 epistasis pathway, where Rad51p binds first, followed by Rad52p, group, defined by the yeast RAD50, RAD51, RAD52, Rad55p, and finally Rad54p. Each of these Rad proteins RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2 genes. also associates with the homologous donor sequences These genes are highly conserved among all eukaryotes during strand invasion. We further examined the func-(Cromie et al., 2001; Pâ ques and Haber, 1999; Sung et tional interdependencies among these proteins by peral., 2000), highlighting the importance of these proteins
Nucleic Acids Research, 1994
Single strand and double strand DNA damage-induced recombination were compared in the yeast Saccharomyces cerevisiae. The non-replicating plasmid pUC1 8-HIS3 was damaged in vitro and introduced into yeast cells; plasmid -chromosome recombinants were selected as stable His+ transformants. Single strand damage was produced by UV irradiation at 254 nm or by psoralen photoreaction at 390 nm. Double strand damage was produced by psoralen photoreaction at 350 nm or by restriction endonuclease digestion. Recombinants were classified as resulting from gene conversion without crossing over, single plasmid integration, or multiple plasmid integration. Single and double strand DNA damage produced different patterns of recombination. In repair proficient cells double strand damage induced primarily multiple plasmid integrations, while single strand damage induced higher proportions of gene conversions and single integrations. Reciprocal recombination depended on the RAD1 gene, which is involved in both excision repair and recombination; plasmid integration induced by all forms of damage was decreased in a radl disruption strain. Mutation of the RAD3 excision repair gene decreased plasmid integration induced by far UV irradiation and psoralen crosslinks, but not by double strand breaks, which are not substrates of nucleotide excision repair. Double strand break-induced plasmid integration was also decreased by disruption of RAD10, which forms a complex with RAD1; disruption of RAD4 had no effect. Thus, while nucleotide excision repair genes are involved in the processing of damaged DNA to generate recombination intermediates, RADI and RAD10 are additionally involved in reciprocal exchange.
… and cellular biology, 1994
In haploid rad52 Saccharomyces cerevisiae strains unable to undergo homologous recombination, a chromosomal double-strand break (DSB) can be repaired by imprecise rejoining of the broken chromosome ends. We have used two different strategies to generate broken chromosomes: (i) a site-specific DSB generated at the AMT locus by HO endonuclease cutting or (ii) a random DSB generated by mechanical rupture during mitotic segregation of a conditionally dicentric chromosome. Broken chromosomes were repaired by deletions that were highly variable in size, all of which removed more sequences than was required either to prevent subsequent HO cleavage or to eliminate a functional centromere, respectively. The junction of the deletions frequently occurred where complementary strands from the flanking DNA could anneal to form 1 to 5 bp, although 12% (4 of 34) of the events appear to have occurred by blunt-end ligation. These types of deletions are very similar to the junctions observed in the repair of DSBs by mammalian cells (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986). When a high level of HO endonuclease, expressed in all phases of the cell cycle, was used to create DSBs, we also recovered a large class of very small (2or 3-bp) insertions in the HO cleavage site. These insertions appear to represent still another mechanism of DSB repair, apparently by annealing and ifiling in the overhanging 3' ends of the cleavage site. These types of events have also been well documented for vertebrate cells.
Molecular and Cellular Biology, 2002
Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51-and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase.
Genetics
We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast. A 1 17-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed. The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid. In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time. The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%). In addition, the fraction of plasmids lost was significantly greater in the inverted orientation. The kinetics of appearance of intermediates and final products were also monitored. The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair. Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics. For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product. We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.
Genetics, 2001
The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is...
Journal of Biological Chemistry, 1996
Saccharomyces cerevisiae rad1 and rad10 mutants are unable to carry out nucleotide excision repair and are also defective in a mitotic intrachromosomal recombination pathway. The products of these genes are subunits of an endonuclease which recognizes DNA duplex/ single-strand junctions and specifically cleaves the 3 single-strand extension at or near the junction. It has been suggested that such junctions arise as a consequence of DNA lesion processing during nucleotide excision repair and the processing of double-strand breaks during intrachromosomal recombination. In this study we show that the RAD1⅐RAD10 complex also cleaves a more complex junction structure consisting of a duplex with a protruding 3 single-strand branch that resembles putative recombination intermediates in the RAD1⅐RAD10-mediated single-strand annealing pathway of mitotic recombination. Using monoclonal antibodies, we have identified two regions of RAD1 that are required for the cleavage of duplex/single-strand junctions. These reagents also inhibit nucleotide excision repair in vitro, confirming the essential role of the RAD1⅐RAD10 endonuclease in this pathway.